From 10-01-06 to 9-01-07, we continued our studies of CTCF - a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of chromatin-insulator function and gene imprinting. Although many of these functions rely on CTCF-DNA interactions, there is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. We demonstrated that a subpopulation of CTCF in chromatin interacts directly with the RNA polymerase II (Pol II) complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly co-localizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Double" ChIP analyses revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin-synthesizing cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-Chip hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. Next, we performed a genome-wide characterization of CTCF sites in human genome. We were able to describe 13,804 novel CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. These results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of a particular class of CTCF sites. Also, these data raise an important question about the overall validity of the ChIP-to-chip approach for CTCF, because many of CTCF-binding sites, especially those in gene-promoters, have been certainly missed by using it. Apparently, the main problems are associated with: 1) the type of anti-CTCF antibodies (because different classes of CTCF-DNA-complexes in chromatin are differentially masked by interactions with different co-factors); 2) method of cross-linking CTCF to DNA before the IP step (because apparently only high-affinity CTCF-sites have been detected and such sites are commonly seen in insulators but not in gene-promoters); and 3) ChIPed-probe-amplification methods (because many CTCF-sites are found to be embedded into extremely CT- and CG-rich sequences that are very difficult, if not possible at all, to PCR by using Taq polymerase). We also continued in depth characterization of the role of CTCF in regulation of several genomic loci that we study in the lab. First, we reported a peculiar interplay of CTCF repression of hTERT gene and methylation of hTERT 5 regulatory region. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. Now we used electrophoretic mobility shift assay (EMSA) and ChIP to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression. Second, while studying regulation by CTCF of imprinted loci we performed systematic chromosome conformation capture 4C analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF site within Igf2 DMR1. This systematic 4C analysis of an imprinted gene-cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome. While studying maternally imprinted KvDMR1 locus we characterized two novel CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated allele. Using a number of reporter assays, we showed that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Next, we studied function of another group of CTCF-binding sites mapped in the Wilms tumor 1 gene (WT1) and characterized the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for CTCF. Potentiation of the silencer activity was demonstrated after CTCF protein was knocked-down, thereby suggesting a novel silencer-blocking activity for CTCF. We also assessed the ARR methylation in developmental and in tumor tissues including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumorigenesis. Finally for this FY report, we discovered the presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters. We suggested (based on the analogy to CTCF/BORIS-sites mapped and functionally characterized in MAGE-A1 and NY-ESO-1 gene promoter-regulation, as we published earlier) that their activation in spermatogenesis is mediated by the programmed replacement of the DNA-occupancy in vivo from CTCF to occupancy of the same site by BORIS. Finally, this year we reported cloning of CTCF from Danio rerio - a valuable vertebrate model organism. It shows very high similarity with CTCF from mammals as well as similar developmental expression pattern and promoter regulation. Taken together with our earlier report on identification and cloning of Drosophila CTCF (H. Moon, et.al., EMBO R., 2005, v.6, pp. 165-170), this work put forward our continuing efforts (which begun in early 90s from cloning of CTCF from birds, mice, rats, and humans) to identify and molecularly clone CTCF from all species that have this gene. To this end, we have identified (but not yet published) a cDNA sequence of CTCF from C. elegans.